The present invention relates to an apparatus for analyzing the nature of particles by causing them to flow, and is particularly concerned with an apparatus efficient for an analysis of flowing cells such as the classification of leukocytes.
An example in which the above-noted particles are cells will be taken in the description below.
One of the conventional methods of distinguishing sample particles in a suspension is flow cytometry. In this method, cells in a suspension, typically a blood sample, are run through the minute passage of a flow cell, where each particle in the blood sample is illuminated by one or more beams of light in the sensing zone. Then, the result of the interaction of a light beam or a group of light beams on each particle is sensed by one or more light sensors. Interaction here means a phenomenon such as the occurrence of fluorescence from particles caused by light beams scattered by particles. Conventionally, light sensors are designed so that they can measure scattered light at a particular scattering angle or fluorescence having a particular wavelength. In this way, each particle flowing through the flow cell is characterized by scattered light, fluorescence or one or more other optical or electrical characteristics. According to these characteristics, each particle is mapped in a specific space based on radiation intensity or another characteristics such as red or green which is measured by the sensor. It has been desired to map different kinds of particles in a sample in correspondingly different areas in the specific space so that the type of each particle (e.g. cell) can be estimated from their mapping in the specific space. Therefore, to improve accuracy of the analysis of particles, the sheath flow method has been employed for causing flow in the flow cell. In this method, a blood sample flows only in the center of the minute passage. Furthermore, a light beam of a substantially elliptical shape whose major axis is perpendicular to the direction of the flow in the flow cell is used so that the quantity of light each particle receives can be uniform. Japanese Unexamined Patent Application Publication (Kokai) No. 63 -47635 discloses means for scanning a laser beam perpendicularly to the direction of the stream in the flow cell in order to expand the area in which the quantity of light is uniform.
As the rate of the flow of a sample increases, the position of each cell flowing through the flow cell varies more greatly. Unless the quantity of laser light is uniform in the area where particles pass, the analysis will be less accurate, because the intensity of scattered light or fluorescence from particles depends on the quantity of laser light. The quantity of laser light in the area where particles pass needs to be uniform. The laser for use in flow cytometry produces a Gaussian beam whose quantity of light is large in the center and small in the periphery.
The above-mentioned conventional techniques have enlarged the width of a laser beam into an elliptical shape to use the center where the quantity of the light is uniform. Such a laser beam has caused energy loss, so a powerful, and thus large, laser has been necessary for these techniques. However, a large laser leads to a large size of the entire equipment which costs much more. Moreover, these techniques have drawbacks such as the heat and vibration from lasers which lower their reliability and shorten their life. Besides, the section of the sample flow in the flow cell must be small because of the narrow range where the quantity of light is uniform. This has prevented the flow of the sample from being enlarged, necessitating a long period of time for analysis.
When a laser beam perpendicular to the direction of the stream in the flow cell, it is necessary to apply a uniform radiation intensity of the laser to the range where cells move during laser scanning. The laser beam must be expanded in the direction of the stream in the flow cell, which leads to a large loss of laser energy.